Electrophoresis is a process for separation of charged species. It is based on the fact that various species migrate with different mobility in an electric field. Small species, like metal ions, as well as large species such as viruses have been separated by electrophoretic techniques. Nevertheless the technique is currently used mostly for separation of biological macromolecules, including proteins, nucleic acids and their derivatives. The process is usually carried out by forcing the molecules to migrate through an aqueous gel. The mobilities depend on structure of the gel, electric field and characteristics of ions themselves, including net surface charge, size and shape.
The gels used in electrophoresis fall into two broad categories. They may be composed of natural or synthetic polymers. Agarose is the most widely used natural material and polyacrylamide gels represent the most common synthetic matrix. The gels are employed in essentially two types of electrophoretic units: vertical and horizontal ones. Running the gels in the vertical format has some advantages. Since the gels are cast between two plates they are uniform, may be very thin and therefore the heat produced during the run can be dissipated easily, resulting in highly reproducible band patterns. On the other hand, it is more difficult to prepare vertical gels and they are prone to tearing during removal of the gel from the cassette. In contrast to vertical units, handling of gels is easy in horizontal electrophoresis units. During a run the gel rests flat on a platform. A contact between the electrodes and the gel may be established directly or by means of wicks. Alternatively, in submerged gel electrophoresis the gel is immersed in buffer which serves as a conductive medium between electrodes and the gel. This format is the simplest and is widely used for analysis of nucleic acids. Agarose gels are almost exclusively used for submerged gel electrophoresis of nucleic acids.
An apparatus for submerged gel electrophoresis usually includes a base and a pair of opposing tanks. Each tank has a buffer containment zone and an electrode. A gel is placed on said base between the tanks and is in communication with the buffer containment zones. The layer of buffer is usually 2-4 mm high above the gel and up to 10 cm high in the tanks. The electrodes are situated typically near the bottom of the tanks parallel to the gel.
Modified versions of the above described apparatus, such as the one described by Turre et al (U.S. Pat. No. 4,415,418) are also known. A similar unit but suitable for bidimensional electrophoresis has been also disclosed (Serwer, U.S. Pat. No. 4,693,804). In these apparatus the electric field density is higher in the gel compartment than in the buffer tanks.
The resolution of nucleic acids in agarose gels using the above described submerged gel electrophoresis units is satisfactory, as it is widely known. When electrophoretic runs need to be performed for a prolonged time it is advantageous to recirculate and cool the buffer. Some submerged gel electrophoresis units, essentially constructed as described above, have a port in each tank. Tubing is then connected to the ports and a pump is used to circulate the buffer. The buffer in the tubing is electrically charged and therefore circulation of buffer in this manner represents a potential safety hazard. In addition, the evenness of buffer flow over the gel surface is not addressed in these units.
An apparatus in which the buffer circulates in another way is Hoefer model HE 100 B (Hoefer Scientific Instruments, San Francisco). Although electrically charged, the buffer circulates inside the unit through a spiral containing a magnetic stirring bar in the center. The spiral is positioned between two buffer tanks. Rotation of the stirring bar by means of a magnetic stirrer on which the electrophoresis unit rests draws the buffer from one tank into the other. However, the electric field density also in this unit is higher in the gel compartment than in the buffer tanks.
Separation of large DNA molecules in agarose gels can be greatly improved by pulsed field electrophoresis (Cantor and Schwartz, U.S. Pat. No. 4,473,452). Other units for pulsed field electrophoresis have been also described (for example, Chu et al (1986) Science, 232, 1582). In some of these units the side and end walls do not define the electric field as in the units described above. The electric field is instead closed by a specific electrode arrangement.
As already noted, polyacrylamide and agarose gels have been the matrices mostly used for electrophoretic analysis of biomolecules. A new synthetic matrix has been introduced for analysis of proteins and nucleic acids by Kozulic et al (U.S. patent application Ser. No. 328,123, Analytical Biochemistry 163 (1987) 506-512 and Analytical Biochemistry 170 (1988) 478-484). It is based on an acrylic monomer, N-acryloyl-tris(hydroxymethyl)aminomethane (NAT). The poly(NAT) gels were found to be more porous than polyacrylamide gels but less porous than agarose gels. Therefore they offer advantages for separation of large proteins and those nucleic acids whose size is out of the optimal separation range of agarose and polyacrylamide gels. In the cited references, the superior properties of the poly(NAT) gels for analysis of DNA were demonstrated after running the gels in a vertical format. However, we have surprisingly found that in the standard submerged electrophoresis units the resolution of DNA in the poly(NAT) gels was never so good as in the vertical system. The major difference was observed in the lower half of the gel, where the bands became much more diffuse. Moreover, the DNA fragments in the middle lanes migrated further than the corresponding fragments in the outer lanes. This phenomenon is known as the smiling effect. Further, very often DNA bands were straight only in the middle but the edges were bent upwards.
The occurrences described above were less pronounced with agarose gels run under identical conditions. Agarose gels used for DNA analysis usually contain from 0.5 to 2% of polymer dry weight. On the contrary, the poly(NAT) gels we have used contained from 6 to 12% of polymer dry weight. It is expected therefore that poly(NAT) gels give a higher resistance to migration of buffer ions through the gel. Accordingly the heat produced during electrophoresis, known as Joule heat, is expectedly higher in poly(NAT) than in agarose gels. There are many examples in prior art demonstrating that controlling Joule heat is essential for achievement of optimal electrophoretic separations. It is also known that the electric field should be as uniform as possible.
In the standard submerged gel electrophoresis apparatus, there is no control over the Joule heat and the electric field is not uniform, as schematically depicted in FIG. 1a. The field density is higher in the gel region 102 than in the buffer tanks 101. In addition, the electric field lines 105 are curved in the gel region. The curvature depends mostly on distance between the electrodes 106, 106' and gel region, position of the electrodes and level of buffer 104 above the gel 103. The curvature can be reduced and the uniformity of electric field improved by placing the electrodes 107, 107' in the same plane to the gel, as shown in FIG. 1b. However, the electric field lines are still curved, especially if the level of buffer is high above the gel. Other positions of the pair of electrodes relative to the gel are possible, but the electric field uniformity cannot be substantially improved. As an element of this invention, it was realized that two pairs of electrodes 108, 108', one positioned above the other, will produce a substantially more uniform electric field in the region between the electrodes, as shown in FIG. 1c. While the uniformity of electric field is very important, it will give the expected improvement in electrophoretic resolution only with an apparatus in which additional requirements are fulfilled. Most important of these additional requirements are control of the Joule heat and prevention of buffer ion depletion in the gel region, that is accumulation of cations close to cathode and anions close to anode. These requirements are fulfilled in the apparatus of the present invention, as will be realized from the explanations hereinunder.